Methods and compositions for treating hemophilia b

ABSTRACT

Disclosed herein are methods and compositions for insertion of Factor IX (FIX) sequences into the genome of a cell for treating hemophilia B.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/392,333, filed Oct. 12, 2010, the disclosure of whichis hereby incorporated by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of gene modification andtreatment of hemophilia.

BACKGROUND

Hemophilia B is a genetic disorder of the blood-clotting system,characterized by bleeding into joints and soft tissues, and by excessivebleeding into any site experiencing trauma or undergoing surgery. Whilehemophilia B is clinically indistinguishable from hemophilia A, factorVIII (FVIII) is deficient or absent in hemophilia A and factor IX (FIXor F.IX) is deficient or absent in patients with hemophilia B. Factor IXencodes one of the serine proteases involved with the coagulationsystem, and it has been shown that restoration of even 3% of normalcirculating levels of wild type Factor IX protein can preventspontaneous bleeding.

Gene therapy, including liver-directed gene therapy protocols and directintramuscular injection, involving the introduction of plasmid and othervectors (e.g., AAV) encoding a functional FIX protein have beendescribed for treatment of hemophilia B. See, e.g., U.S. Pat. No.6,936,243; Lee et al. (2004) Pharm. Res. 7:1229-1232; Graham et al.(2008) Genet Vaccines Ther. 3:6-9. However, in these protocols, theformation of inhibitory anti-factor IX (anti-FIX) antibodies andantibodies against the delivery vehicle remains a major complication ofFIX protein replacement-based treatment for hemophilia B.

U.S. patent application Ser. No. 12/798,749 describes targetedintegration of a functional FIX protein into isolated stem cells andtreatment of hemophilia B by introduction of the FIX-producing stemcells into patients in need of treatment.

However, there remains a need for additional compositions and methods oftreating hemophilia B in subjects with this disease.

SUMMARY

Disclosed herein are methods and compositions for targeted integrationof a sequence encoding a functional FIX protein to treat hemophilia B.In particular, the methods involve administering nucleases that mediatetargeted insertion of sequence encoding a functional FIX protein intothe genome of cells for amelioration of the disease.

In one aspect, described herein is a DNA binding domain (e.g.,zinc-finger protein (ZFP) or TALE protein) that binds to target site ina region of interest (e.g., an Factor IX gene) in a genome, wherein theZFP comprises one or more engineered zinc-finger binding domains and theTALE comprises one or more engineered TALE DNA-binding domains. In oneembodiment, the DNA binding domain is a nuclease, e.g., a ZFP is azinc-finger nuclease (ZFN) and a TALE is a TALE nuclease (TALEN) thatcleaves a target genomic region of interest, wherein the ZFN or TALENcomprises one or more engineered DNA binding domains and a nucleasecleavage domain or cleavage half-domain. Cleavage domains and cleavagehalf domains can be obtained, for example, from various restrictionendonucleases and/or homing endonucleases. In one embodiment, thecleavage half-domains are derived from a Type IIS restrictionendonuclease (e.g., Fok I). In certain embodiments, the zinc fingerdomain recognizes a target site in an endogenous FIX gene for example azinc finger domain as shown in Table 1 (or a zinc finger domain thatbinds to a target site as shown in Table 1).

In another aspect, described herein is a polynucleotide encoding one ormore ZFNs and/or TALENs described herein. The polynucleotide may be, forexample, mRNA.

In another aspect, described herein is a ZFN and/or TALEN expressionvector comprising a polynucleotide, encoding one or more ZFNs and/orTALENs described herein, operably linked to a promoter. In oneembodiment, the expression vector is a viral vector. In one aspect, theviral vector exhibits tissue specific tropism.

In another aspect, described herein is a host cell comprising one ormore ZFN and/or TALEN expression vectors. The host cell may be stablytransformed or transiently transfected or a combination thereof with oneor more ZFP or TALEN expression vectors. In one embodiment, the hostcell is an embryonic stem cell. In other embodiments, the one or moreZFP and/or TALEN expression vectors express one or more ZFNs and/orTALENs in the host cell. In another embodiment, the host cell mayfurther comprise an exogenous polynucleotide donor sequence. In any ofthe embodiments, described herein, the host cell can comprise a livercell, a muscle cell, a stem cell or an embryo cell. The cells may befrom any organism, for example human, non-human primate, mouse, rat,rabbit, cat, dog or other mammalian cells.

In another aspect, provided herein are methods for treating hemophilia Busing nucleases to integrate a sequence encoding a FIX protein in a cellin a subject in need thereof. In certain embodiments, the FIX-encodingsequence is delivered using a viral vector, a non-viral vector (e.g.,plasmid) and/or combinations thereof. In certain embodiments, the vectorcomprises an AAV vector, such as AAV8. In certain embodiments, thenucleases and/or FIX-encoding sequences are delivered via intravenous(e.g., intra-portal vein) administration into the liver of an intactanimal.

In any of the methods described herein, the nuclease can be one or morezinc finger nucleases, one or more homing endonucleases (meganucleases)and/or one or more TAL-effector domain nucleases (“TALEN”). Thenucleases (e.g., ZFN and/or TALEN) as described herein may bind toand/or cleave the region of interest in a coding or non-coding regionwithin or adjacent to the gene, such as, for example, a leader sequence,trailer sequence or intron, or within a non-transcribed region, eitherupstream or downstream of the coding region. In certain embodiments, theZFN binds to and/or cleaves an endogenous Factor IX gene (mutant orwild-type). In other embodiments, the ZFN and/or TALEN binds to and/orcleaves a safe-harbor gene (e.g., any gene which disruption of is nottoxic or disruptive to the cell), for example a CCR5 gene, a PPP1R12c(also known as AAV S1) gene or a Rosa gene. See, e.g., U.S. PatentPublication Nos. 20080299580; 20080159996 and 201000218264.

Furthermore, any of the methods described herein may further compriseadditional steps, including partial hepatectomy or treatment withsecondary agents that enhance transduction and/or induce hepatic cellsto undergo cell cycling. Examples of secondary agents include gammairradiation, UV irradiation, tritiated nucleotides such as thymidine,cis-platinum, etoposide, hydroxyurea, aphidicolin, prednisolone, carbontetrachloride and/or adenovirus.

The methods described herein can be practiced in vitro, ex vivo or invivo. In certain embodiments, the compositions are introduced into alive, intact mammal. The mammal may be at any stage of development atthe time of delivery, e.g., embryonic, fetal, neonatal, infantile,juvenile or adult. Additionally, targeted cells may be healthy ordiseased. In certain embodiments, the compositions (e.g.,polynucleotides encoding nuclease(s) and/or FIX-encoding sequences) aredelivered to the liver of a live animal, for example via intraportalinjection. In other embodiments, one or more of the compositions aredelivered intravenously (other than the intraportal vein, for exampletail vein injection), intra-arterially, intraperitoneally, into liverparenchyma (e.g., via injection), into the hepatic artery (e.g., viainjection), and/or through the biliary tree (e.g., via injection)

For targeting the compositions to a particular type of cell, e.g.,hepatocytes, one or more of the administered compositions may beassociated with a homing agent that binds specifically to a surfacereceptor of the cell. For example, the vector may be conjugated to aligand (e.g., galactose) for which certain hepatic system cells havereceptors. The conjugation may be covalent, e.g., a crosslinking agentsuch as glutaraldehyde, or noncovalent, e.g., the binding of anavidinated ligand to a biotinylated vector. Another form of covalentconjugation is provided by engineering the AAV helper plasmid used toprepare the vector stock so that one or more of the encoded coatproteins is a hybrid of a native AAV coat protein and a peptide orprotein ligand, such that the ligand is exposed on the surface of theviral particle.

The target cells may be human cells, or cells of other mammals(including veterinary animals), especially nonhuman primates and mammalsof the orders Rodenta (mice, rats, hamsters), Lagomorpha (rabbits),Carnivora (cats, dogs), and Arteriodactyla (cows, pigs, sheep, goats,horses). In some aspects, the target cells comprise a tissue (e.g.liver). In some aspects, the target cell is a stem cell (e.g., anembryonic stem cell, an induced pluripotent stem cell, a hepatic stemcell, etc.) or animal embryo by any of the methods described herein, andthen the embryo is implanted such that a live animal is born. The animalis then raised to sexual maturity and allowed to produce offspringwherein at least some of the offspring comprise the genomicmodification.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole. Thus, the disclosure encompasses thefollowing embodiments:

1. A protein comprising an engineered zinc finger protein DNA-bindingdomain, wherein the DNA-binding domain comprises four or five zincfinger recognition regions ordered F1 to F4 or F1 to F5 from N-terminusto C-terminus, and wherein

(i) when the DNA-binding domain comprises five zinc finger recognitionregions, F1 to F5 comprise the following amino acid sequences:

(SEQ ID NO: 4) F1: QSGDLTR (SEQ ID NO: 5) F2: RSDVLSE (SEQ ID NO: 6)F3: DRSNRIK (SEQ ID NO: 7) F4: RSDNLSE (SEQ ID NO: 8) F5: QNATRIN;

(ii) when the DNA-binding domain comprises four zinc finger recognitionregions, F1 to F4 comprise the following amino acid sequences:

(SEQ ID NO: 10) F1: RSDSLSV (SEQ ID NO: 11) F2: TSGHLSR (SEQ ID NO: 12)F3: RSDHLSQ (SEQ ID NO: 13) F4: HASTRHC.

2. The protein according to 1, further comprising a cleavage domain orcleavage half-domain.

3. The protein of 2, wherein the cleavage half-domain is a wild-type orengineered FokI cleavage half-domain.

4. A polynucleotide encoding the protein of any of 1 to 3.

5. A gene delivery vector comprising a polynucleotide of 4.

6. An isolated cell comprising the protein of any of 1 to 3 or thepolynucleotide of 4.

7. An isolated cell comprising the protein of any of the 1 to 3 or thepolynucleotide of 4.

8. A method for treating hemophilia B in a subject, the methodcomprising inserting (e.g., via targeted integration) a sequenceencoding a functional Factor IX (FIX) protein into the genome of a cellusing at least one nuclease, wherein the subject comprises the cell.

9. The method of 8, wherein the sequence is integrated into anendogenous gene.

10. The method of 9, wherein the endogenous gene is selected from thegroup consisting of a FIX gene and a safe-harbor gene.

11. The method of any of 8 to 10, wherein the sequence and/or thenuclease is delivered to the cell using a vector selected from the groupconsisting of a viral vector, a non-viral vector and combinationsthereof.

12. The method of any of 8 to 11, wherein the cell is a hepatic cell andthe sequence is delivered to the cell by intravenous administration(e.g., into the liver) of an intact animal.

13. The method of any of 8 to 12, wherein the at least one nuclease is azinc finger nuclease, a TALEN or a homing endonuclease.

14. The method of any of 8 to 13, further comprising the step ofperforming a partial hepatectomy on the subject.

15. The method of any of 8 to 14, further comprising the step oftreating the subject with at least one secondary agent.

16. The method of 15, wherein the secondary agent is selected from thegroup consisting of gamma irradiation, UV irradiation, tritiatednucleotides, cis-platinum, prednisolone, carbon tetrachloride,etoposide, hydroxyurea, aphidicolin, adenovirus and combinationsthereof.

17. The method of any of 8 to 16, wherein the cell is an isolated celland the method further comprises administering the isolated cell to thesubject.

18. The method of any of 8 to 17, wherein the subject is selected fromthe group consisting of an embryo, a fetus, a neonate, an infant, ajuvenile or an adult.

19. The method of any of 8 to 18, further comprising associating thesequence with a homing agent that binds specifically to a surfacereceptor of the cell.

20. The method of 19, wherein the homing agent comprises galactose or ahybrid of an AAV coat protein and galactose.

21. The method of any of 8 to 20, further comprising associating apolynucleotide encoding the at least one nuclease with a homing agentthat binds specifically to a surface receptor of the cell.

22. The method of 21, wherein the homing agent comprises galactose or ahybrid of an AAV coat protein and galactose.

23. The method of any of 8 to 22, wherein the cell is selected from thegroup consisting of a human cell, a nonhuman primate cell, a Rodentacell, a Lagomorpha cell, a Carnivora cell and an Arteriodactyla cell.

24. The method of any of 8 to 22, wherein the target cell is a stemcell.

25. The method of 24, wherein the stem cell is an embryonic stem cell,an induced pluripotent stem cell, a hematopoietic stem cell, ahepatocyte or a hepatic stem cell.

26. The method of any of 8 to 25, wherein the nuclease comprises a zincfinger nuclease according to any of 1 to 3, a polynucleotide accordingto 4 or a gene delivery vehicle according to 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panels A-E, show N2 ZFNs efficiently cleave the gene encodingFactor IX (F9) intron 1 and induce homologous recombination in humancells. FIG. 1A depicts a schematic depicting the target of the N2 ZFNpair in intron 1 of the human F9 gene. FIG. 1B depicts the bi-cistronicFLAG-tagged ZFN expression plasmid. FIG. 1C shows a gel with the resultsof a Surveyor® mismatch assay (Transgenomics, “Cel-I”) followingtransfection of the N2 ZFN expression plasmid into K562 cells. The assaydemonstrates the result of NHEJ repair of a DSB induced by the N2 ZFNsat intron 1 of the hF9 gene at day 3 post-transfection. ZFN expressionwas confirmed by α-FLAG immunoblotting and protein loading was assessedusing an α-NFkB p65 antibody. FIG. 1D shows a schematic of the targetedintegration (TI) assay detailing the time-course of ZFN-mediatedtargeting of a NheI restriction site tag into the hF9 gene. FIG. 1Eshows a gel depicting the results of a RFLP assay followingco-transfection of ZFN expression plasmid with increasing amounts ofNheI tag donor plasmid (0-4 μg). The data show increasing levels of genetargeting at days 3 and 10 post-transfection, whereas transfection ofthe NheI tag donor alone (4 μg, ‘(−) ZFN’) does not result in detectablegene targeting. Black arrows denote NheI-sensitive cleavage productsresulting from TI at both day 3 and 10. TI PCR performed with PCR using³²P-labeled nucleotides and the band intensity was quantified byphosphorimager. ZFN expression confirmed by α-FLAG immunoblotting andprotein loading was assessed using an α-NFkB p65 antibody.

FIG. 2, panels A-E, show AAV-mediated delivery of N2 ZFNs to miceincluding an N2 “landing pad” (LP) results in efficient cleavage of theLanding Pad (LP) intron 1. FIG. 2A depicts a diagram showing how the N2ZFNs target intron 1 of a human F9 mini-gene (LP), which mimics apublished HB-causing mutation (Thompson et al, (1994) Hum. Genet. 94:299-302). FIG. 2B shows the gel from a PCR analysis demonstrating the LPconstruct has been knocked in to the mouse ROSA26 locus. FIG. 2C showsthe results of an ELISA to detect circulating plasma hFIX. The datashows that LP mice do not have circulating plasma hFIX, as measuredusing a hFIX-specific ELISA, unless the mice are injected with a viralvector expressing hFIX (1e10 viral genomes (v.g.) AAV-hFIX injected viatail vein). FIG. 2D depicts a bi-cistronic AAV8-N2 ZFN expression vectorwith expression controlled by the ApoE enhancer and humanalpha1-antitrypsin promoter. FIG. 2E shows the results of a Cel-I assayperformed following tail vein injection of 1e11 v.g. AAV-N2 expressionvector into LP mice that results in cleavage of the LP intron 1 in liverDNA at day 7 post-injection. Cel-1 assay was performed with a PCRamplicon using ³²P-labeled nucleotides and the band intensity quantifiedby phosphorimager. ZFN expression is confirmed by α-FLAG immunoblottingof whole liver lysates and protein loading was assessed using an α-NFkBp65 antibody.

FIG. 3, panels A and B, show N2 ZFNs promote AAV-mediated targeting ofwild-type F9 exons 2-8 to Landing Pad intron 1 in vivo. FIG. 3A shows aschematic of how the LP gene mutation can be bypassed by TI of hF9 exons2-8 into intron 1. Targeted and untargeted LP alleles can bedifferentiated through PCR using primers P1, P2, and P3. FIG. 3B depictsa gel of a PCR analysis with primer pairs P1/P2 and P1/P3 demonstratingsuccessful gene targeting upon I.P. co-injection of 5e10 v.g. AAV8-N2and 2.5e11 v.g. AAV8-Donor in LP/HB mice at day 2 of life, but not withinjection of 5e10 v.g. AAV8-N2 alone, or co-injection of 5e10 v.g.AAV8-Mock and 2.5e11 v.g. AAV8-Donor. PCR was performed using³²P-labeled nucleotides, allowing for quantification of product bandintensity by phosphorimager to evaluate targeting frequency. In targetedsamples, primers P1 and P2 will generate a smaller product indicatingsuccessful amplification of the targeted wild-type F9 exons 2-8, whileprimers P1 and P3 will generate a larger product than the untargetedallele.

FIG. 4, panels A-F, show in vivo hepatic gene correction results intherapeutic levels of circulating FIX. FIG. 4A is a graph showing theplasma hFIX levels in LP mice following I.P. injection at day 2 of lifewith either 5e10 v.g. AAV-N2 alone (n=7 pre- and post-partialhepatectomy (PHx)), 5e10 v.g. AAV-N2 and 2.5e11 v.g. AAV-Donor (n=7 pre-and post-PHx), or 5e10 v.g. AAV-Mock and 2.5e11 v.g. AAV-Donor (n=6 pre-and post-PHx). Timing of the PHx is indicated by the arrow. Error barsdenote standard error. FIG. 4B is a graph showing plasma hFIX levels inwild-type mice (n=5) following tail vein injection of 1e12 v.g. AAV-hFIX(predominantly episomal) with subsequent PHx. Error bars denote standarderror. FIG. 4C is a graph showing plasma hFIX levels in wild-type micefollowing I.P. injection at day 2 of life with either 5e10 v.g. AAV-N2alone (n=8 pre-PHx, n=4 post-PHx), 5e10 v.g. AAV-N2 and 2.5e11 v.g.AAV-Donor (n=9 pre-PHx, n=5 post-PHx), or 5e10 v.g. AAV-Mock and 2.5e11v.g. AAV-Donor (n=6 pre-PHx, n=5 post-PHx). Error bars denote standarderror. FIG. 4D is a graph of plasma hFIX levels in LP/HB mice followingintraperitoneal (I.P.) injection at day 2 of life with either 5e10 v.g.AAV-N2 alone (n=10 pre-PHx, n=1 post-PHx), 5e10 v.g. AAV-N2 and 2.5e11v.g. AAV-Donor (n=9 pre-PHx, n=5 post-PHx), or 5e10 v.g. AAV-Mock and2.5e11 v.g. AAV-Donor (n=9 pre-PHx, n=3 post-PHx). Error bars denotestandard error. FIG. 4E shows a gel demonstrating liver-specificexpression of hFIX RNA as detected by RT-PCR at week 20 of life in anLP/HB mouse receiving I.P. injection at day 2 of life with 5e10 v.g.AAV-N2 and 2.5e11 v.g. AAV-Donor. FIG. 4F is a graph showing the time toclot formation as assayed by the activated partial thromboplastin time(aPTT) assay at week 14 of life of mice receiving LP injection at day 2of life with 5e10 v.g. AAV-N2 and 2.5e11 v.g. AAV-Donor (n=5), or 5e10v.g. AAV-Mock and 2.5e11 v.g. AAV-Donor (n=3) (p-value from 2-tailedStudent's t-test). aPTTs of wild-type (WT) and hemophilia B (HB) miceare shown for comparison.

FIG. 5, panels A and B, show in vivo hepatic gene correction results inthe expression of therapeutic levels of circulating FIX. FIG. 5A is agraph showing the plasma hFIX levels in adult LP mice following I.V.injection at 6 weeks of age with either 1e¹¹ v.g./mouse AAV-N2 alone(‘ZFN alone’), 1e¹¹ v.g./mouse AAV-N2 and 5.5e11 v.g./mouse AAV-Donor(‘ZFN+Donor’), or 1e¹¹ v.g./mouse AAV-Mock and 5.5e11 v.g. AAV-Donor(‘Mock+Donor’). The data depicted is representative of 3 experimentswith approximately 20 mice per group. In these experiments, the wildtype hF.IX levels were approximately 1000 ng/mL. FIG. 5B is a graphshowing the plasma hFIX levels in adult LP mice following I.V. injectionat 6 weeks of age with either 1e¹¹ v.g./mouse AAV-N2 alone (‘ZFNalone’), 1e¹¹ v.g./mouse AAV-N2 and 5.5e11 v.g./mouse AAV-Donor(‘ZFN+Donor’), or 1e¹¹ v.g./mouse AAV-Mock and 5.5e11 v.g. AAV-Donor(‘Mock+Donor’). Two days following injection, the groups in FIG. 5B weregiven a partial hepatectomy. The data depicted is representative of 3experiments with approximately 20 mice per group. In these experiments,the wild type hF.IX levels were approximately 1000 ng/mL. The datademonstrate that hF.IX expression is stable when given to adult micewith or without a follow on partial hepatectomy, and that it is possibleto perform genome editing in adult animals.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for treating patients withhemophilia B. In particular, nuclease-mediated targeted integration isused to insert a sequence encoding FIX into the genome of one or morecells of the subject (in vivo or ex vivo), such that the cells produceFIX in vivo. In certain embodiments, the methods further compriseinducing cells of the subject, particularly liver cells, to proliferate(enter the cell cycle), for example, by partial hepatectomy and/or byadministration of one or more compounds that induce hepatic cells toundergo cell cycling. Subjects include but are not limited to humans,non-human primates, veterinary animals such as cats, dogs, rabbits,rats, mice, guinea pigs, cows, pigs, horses, goats and the like.

The methods described herein result in treatment of hemophilia B. Unlikepreviously described methods in in vivo models of nuclease-mediated genecorrection using meganucleases (see Gouble et al, (2006) J Gene Med.May; 8(5):616-22) little or no toxicity is observed followingnuclease-mediated integration of a FIX gene in the animal models. Inaddition, the methods and compositions of the invention are functionalin neonates and adult animals, leading to functional activity of theinserted Factor IX transgene.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P.M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. application Ser. No.13/068,735.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197and WO 02/099084.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides, including but not limited to, donor captureby non-homologous end joining (NHEJ) and homologous recombination. Forthe purposes of this disclosure, “homologous recombination (HR)” refersto the specialized form of such exchange that takes place, for example,during repair of double-strand breaks in cells via homology-directedrepair mechanisms. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingerproteins or TALEN can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528 and 2008/0131962,incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogeneous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP as described herein.Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to an activation domain, the ZFP DNA-bindingdomain and the activation domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the activation domain isable to upregulate gene expression. When a fusion polypeptide in which aZFP DNA-binding domain is fused to a cleavage domain, the ZFPDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

A “safe harbor” locus is a locus within the genome wherein a gene may beinserted without any deleterious effects on the host cell. Mostbeneficial is a safe harbor locus in which expression of the insertedgene sequence is not perturbed by any read-through expression fromneighboring genes. Non-limiting examples of safe harbor loci inmammalian cells are the AAVS1 gene (see U.S. Publication No.20080299580), the CCR5 gene (see U.S. Publication No. 20080159996),and/or the Rosa locus (see WO 2010/065123).

Nucleases

Described herein are compositions, particularly nucleases, that areuseful in integration of a sequence encoding a functional FIX protein inthe genome of a cell from a subject with hemophilia B. In certainembodiments, the nuclease is naturally occurring. In other embodiments,the nuclease is non-naturally occurring, i.e., engineered in theDNA-binding domain and/or cleavage domain. For example, the DNA-bindingdomain of a naturally-occurring nuclease may be altered to bind to aselected target site (e.g., a meganuclease that has been engineered tobind to site different than the cognate binding site). In otherembodiments, the nuclease comprises heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; TAL-effector domain DNAbinding proteins; meganuclease DNA-binding domains with heterologouscleavage domains).

A. DNA-Binding Domains

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family andthe HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI,PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences areknown. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue.

In certain embodiments, the nuclease comprises an engineered(non-naturally occurring) homing endonuclease (meganuclease). Therecognition sequences of homing endonucleases and meganucleases such asI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases andmeganucleases may be altered in the context of the nuclease as a whole(i.e., such that the nuclease includes the cognate cleavage domain) ormay be fused to a heterologous cleavage domain.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. patent application Ser. No. 13/068,735,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like (TAL) effectors which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TAL-effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet. 218: 127-136 andWO2010079430). TAL-effectors contain a centralized domain of tandemrepeats, each repeat containing approximately 34 amino acids, which arekey to the DNA binding specificity of these proteins. In addition, theycontain a nuclear localization sequence and an acidic transcriptionalactivation domain (for a review see Schornack S, et al (2006) J PlantPhysiol 163(3): 256-272). In addition, in the phytopathogenic bacteriaRalstonia solanacearum two genes, designated brg11 and hpx17 have beenfound that are homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. See, e.g., U.S. patentapplication Ser. No. 13/068,735, incorporated by reference in itsentirety herein.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal, ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues at positions 12 and 13 withthe identity of the contiguous nucleotides in the TAL-effector's targetsequence (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch etal (2009) Science 326:1509-1512). Experimentally, the natural code forDNA recognition of these TAL-effectors has been determined such that anHD sequence at positions 12 and 13 leads to a binding to cytosine (C),NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds toT. These DNA binding repeats have been assembled into proteins with newcombinations and numbers of repeats, to make artificial transcriptionfactors that are able to interact with new sequences and activate theexpression of a non-endogenous reporter gene in plant cells (Boch et al,ibid). Engineered TAL proteins have been linked to a FokI cleavage halfdomain to yield a TAL effector domain nuclease fusion (TALEN) exhibitingactivity in a yeast reporter assay (plasmid based target). See, e.g.,U.S. patent application Ser. No. 13/068,735; Christian et al((2010)<Genetics epub 10.1534/genetics.110.120717).

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692;6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054;7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos.2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein byreference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Thus, the nuclease comprises a DNA-binding domain in that specificallybinds to a target site in any gene into which it is desired to insert asequence encoding a FIX protein.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease. For example, ZFP DNA-binding domains havebeen fused to nuclease domains to create ZFNs—a functional entity thatis able to recognize its intended nucleic acid target through itsengineered (ZFP) DNA binding domain and cause the DNA to be cut near theZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996)Proc Natl Acad Sci USA 93(3):1156-1160. More recently, ZFNs have beenused for genome modification in a variety of organisms. See, forexample, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014,275. Likewise, TALE DNA-binding domains have beenfused to nuclease domains to create TALENs. See, e.g., U.S. applicationSer. No. 13/068,735.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987;20070305346 and 20080131962, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets forinfluencing dimerization of the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:1538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:1499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type H is (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type H is (H) residue at position 537 with a Lys(K) residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Patent Publication No. 20110201055). Inother embodiments, the engineered cleavage half domain comprises the“Sharkey” and/or “Sharkey'” mutations (see Guo et al, (2010) J. Mol.Biol. 400(1):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962; and 20110201055.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014,275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

Target Sites

As described in detail above, DNA domains can be engineered to bind toany sequence of choice, for example in an endogenous FIX gene or anendogenous or inserted safe-harbor gene. An engineered DNA-bindingdomain can have a novel binding specificity, compared to anaturally-occurring DNA-binding domain. Engineering methods include, butare not limited to, rational design and various types of selection.Rational design includes, for example, using databases comprisingtriplet (or quadruplet) nucleotide sequences and individual zinc fingeramino acid sequences, in which each triplet or quadruplet nucleotidesequence is associated with one or more amino acid sequences of zincfingers which bind the particular triplet or quadruplet sequence. See,for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261,incorporated by reference herein in their entireties. Rational design ofTAL-effector domains can also be performed. See, e.g., U.S. ProvisionalApplication Nos. 61/395,836 and 61/401,429, filed May 17, 2010 and Aug.21, 2010, respectively.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement ofbinding specificity for zinc finger binding domains has been described,for example, in co-owned WO 02/077227.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Application Publication Nos. 20050064474 and 20060188987,incorporated by reference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. application Ser. No. 13/066,735.

For treatment of hemophilia B via targeted insertion of a sequenceencoding a functional FIX protein, any desired site of insertion in thegenome of the subject is cleaved with a nuclease, which stimulatestargeted insertion of the donor polynucleotide carrying the FIX-encodingsequence. DNA-binding domains of the nucleases may be targeted to anydesired site in the genome.

In certain embodiments, the DNA-binding domain of the nuclease istargeted to the endogenous FIX (F9) gene, as described for example inU.S. patent application Ser. No. 12/798,749. The target sites may beanywhere in the coding sequence or upstream or downstream of the codingsequence. In certain embodiments, the target site(s) is (are) near the3′ end of the coding sequence.

In other embodiments, the nuclease (DNA-binding domain component) istargeted to a “safe harbor” locus, which includes, by way of exampleonly, the AAVS1 gene (see U.S. Publication No. 20080299580), the CCR5gene (see U.S. Publication No. 20080159996), and/or the Rosa locus (seeWO 2010/065123).

Donor Sequences

For treating hemophilia, the donor sequence comprises a sequenceencoding a functional FIX protein, or part thereof, to result in asequence encoding and expressing a functional FIX protein followingdonor integration. The donor molecule may be inserted into an endogenousgene such that all, some or none of the endogenous gene is expressed.For example, a transgene comprising function FIX sequences as describedherein may be inserted into an endogenous FIX locus such that some ornone of the endogenous FIX is expressed with the FIX transgene (e.g.,the donor may correct a mutation such that the wild-type endogenoussequences are expressed). In other embodiments, the FIX transgene isintegrated into any endogenous locus, for example a safe-harbor locus(endogenous or inserted). See, e.g., US patent publications 20080299580;20080159996 and 201000218264.

The FIX donor sequence can be introduced into the cell prior to,concurrently with, or subsequent to, expression of the fusionprotein(s). The FIX donor polynucleotide typically contains sufficienthomology to a genomic sequence to support homologous recombination (orhomology-directed repair) between it and the genomic sequence to whichit bears homology. See, e.g., U.S. Patent Publication Nos. 2005/0064474;2007/0134796 and 2009/0263900. It will be readily apparent that thedonor sequence is typically not identical to the genomic sequence thatit replaces. For example, the sequence of the donor polynucleotide cancontain one or more single base changes, insertions, deletions,inversions or rearrangements with respect to the genomic sequence, solong as sufficient homology with chromosomal sequences is present.Alternatively, a donor sequence can contain a non-homologous sequenceflanked by two regions of homology. Additionally, donor sequences cancomprise a vector molecule containing sequences that are not homologousto the region of interest in cellular chromatin. A donor molecule cancontain several, discontinuous regions of homology to cellularchromatin. For example, for targeted insertion of sequences not normallypresent in a region of interest, said sequences can be present in adonor nucleic acid molecule and flanked by regions of homology tosequence in the region of interest.

The FIX donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues. A polynucleotide can be introduced into a cell as part of avector molecule having additional sequences such as, for example,replication origins, promoters and genes encoding antibiotic resistance.Moreover, donor polynucleotides can be introduced as naked nucleic acid,as nucleic acid complexed with an agent such as a liposome or poloxamer,or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus).

The FIX donor is generally inserted so that its expression is driven bythe endogenous promoter at the integration site (e.g., the endogenousFIX promoter when the donor is integrated into the patient's defectiveFIX (F9) locus). However, it will be apparent that the donor maycomprise a promoter and/or enhancer, for example a constitutive promoteror an inducible or tissue specific (e.g., liver specific) promoter thatdrives expression of the function FIX protein upon integration.

The FIX donor sequence can be integrated specifically into any targetsite of choice, thereby eliminating the issues associated with randomintegration in traditional gene therapy. In certain embodiments, thedonor sequence is integrated into the endogenous FIX locus to correctthe deficiency in the patient with hemophilia B. In other embodiments,the FIX donor sequence is integrated into a safe harbor locus, forexample CCR5 locus, AAVS1 locus or the like.

Furthermore, although not required for expression, exogenous sequencesmay also contain transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger protein(s). Any vector systems may be used including, butnot limited to, plasmid vectors, retroviral vectors, lentiviral vectors,adenovirus vectors, poxvirus vectors; herpesvirus vectors andadeno-associated virus vectors, etc. See, also, U.S. Pat. Nos.6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed for treatment. Thus, when one ormore nucleases and a donor construct are introduced into the cell, thenucleases and/or donor polynucleotide may be carried on the same vectoror on different vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple nucleases and/or donorconstructs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of in vivo delivery of engineered DNA-binding proteins and fusionproteins comprising these binding proteins, see, e.g., Rebar (2004)Expert Opinion Invest. Drugs 13(7):829-839; Rossi et al. (2007) NatureBiotech. 25(12):1444-1454 as well as general gene delivery referencessuch as Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiamidet (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh.10 and any novel AAV serotype canalso be used in accordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ12 cells or PA317 cells, which package retrovirus.Viral vectors used in gene therapy are usually generated by a producercell line that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a. receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides (e.g.nuclease-encoding and/or FIX-encoding) described herein includenon-integrating lentivirus vectors (IDLV). See, for example, Ory et al.(1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al. (1998) J.Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol. 72:9873-9880;Follenzi et al. (2000) Nature Genetics 25:217-222; U.S. PatentPublication No 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by a plasmid, while theone or more nucleases can be carried by a AAV vector. Furthermore, thedifferent vectors can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Thus, the instant disclosure includes in vivo or ex vivo treatment ofHemophilia B, via nuclease-mediated integration of FIX-encodingsequence. The compositions are administered to a human patient in anamount effective to obtain the desired concentration of the therapeuticFIX polypeptide in the serum, the liver or the target cells.Administration can be by any means in which the polynucleotides aredelivered to the desired target cells. For example, both in vivo and exvivo methods are contemplated. Intravenous injection to the portal veinis a preferred method of administration. Other in vivo administrationmodes include, for example, direct injection into the lobes of the liveror the biliary duct and intravenous injection distal to the liver,including through the hepatic artery, direct injection in to the liverparenchyma, injection via the hepatic artery, and/or retrogradeinjection through the biliary tree Ex vivo modes of administrationinclude transduction in vitro of resected hepatocytes or other cells ofthe liver, followed by infusion of the transduced, resected hepatocytesback into the portal vasculature, liver parenchyma or biliary tree ofthe human patient, see e.g., Grossman et al., (1994) Nature Genetics,6:335-341.

The effective amount of nuclease(s) and FIX donor to be administeredwill vary from patient to patient and according to the therapeuticpolypeptide of interest. Accordingly, effective amounts are bestdetermined by the physician administering the compositions andappropriate dosages can be determined readily by one of ordinary skillin the art. After allowing sufficient time for integration andexpression (typically 4-15 days, for example), analysis of the serum orother tissue levels of the therapeutic polypeptide and comparison to theinitial level prior to administration will determine whether the amountbeing administered is too low, within the right range or too high.Suitable regimes for initial and subsequent administrations are alsovariable, but are typified by an initial administration followed bysubsequent administrations if necessary. Subsequent administrations maybe administered at variable intervals, ranging from daily to annually toevery several years. One of skill in the art will appreciate thatappropriate immunosuppressive techniques may be recommended to avoidinhibition or blockage of transduction by immunosuppression of thedelivery vectors, see e.g., Vilquin et al., (1995) Human Gene Ther.,6:1391-1401.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN).It will be appreciated that this is for purposes of exemplification onlyand that other nucleases can be used, for instance homing endonucleases(meganucleases) with engineered DNA-binding domains and/or fusions ofnaturally occurring of engineered homing endonucleases (meganucleases)DNA-binding domains and heterologous cleavage domains or TALENs.

EXAMPLES Example 1 Fix Specific ZFNs and their Use for TargetedIntegration

Gene transfer as a strategy for treating genetic disease has beensuccessfully carried out in a variety of animal models of disease, andmore recently, in human applications (see, e.g., Aiuti et al, (2009) N.Engl. J. Med. 360: 447-458; Maguire et al, (2009) N. Engl. J. Med. 358:2240-2248; Cartier et al, (2009) Science 326: 818-823). Gene targetinghas been used to correct ex vivo cultured ES-like induced pluripotentstem cells (Hanna et al, (2007) Science 318: 1920-1923), but themajority of genetic diseases affect organ systems where ex vivomanipulation is currently not feasible. One such organ is the liver, themajor site of plasma protein synthesis, including the blood coagulationfactors. A model genetic disease for liver gene therapy is hemophilia B,caused by deficiency of blood coagulation factor IX (FIX), encoded bythe F9 gene. Targeted integration (TI) of the wild type exons 2-8 intoF9 intron 1 would allow for splicing of wild type coding sequence withexon 1 (FIG. 1A), leading to expression of wild type FIX and rescue ofthe defect caused by most F9 mutations. We thus sought to investigatewhether ZFNs combined with a targeting vector carrying the wild type F9exons 2-8 could induce gene targeting in vivo, to correct a mutated F9gene in situ within the genome of hepatocytes.

ZFN pairs targeting the human F9 intron 1 were used to test the abilityof these ZFNs to induce DSBs at a specific target site. The Cel-I assay(Surveyor™, Transgenomics. Perez et al, (2008) Nat. Biotechnol. 26:808-816 and Guschin et al, (2010) Methods Mol. Biol. 649:247-56), wasused where PCR-amplification of the target site was followed byquantification of insertions and deletions (indels) using the mismatchdetecting enzyme Cel-I (Yang et al, (2000) Biochemistry 39, 3533-3541)which provides a lower-limit estimate of DSB frequency. Three daysfollowing transfection of the ZFN expression vector, genomic DNA wasisolated from K562 cells using the DNeasy kit (Qiagen). Primers for theCel-I analysis of hF9 intron 1 were N2 For (TCGGTGAGTGATTTGCTGAG, SEQ IDNO:1) and N2 Rev (AACCTCTCACCTGGCCTCAT, SEQ ID NO:2). The highestactivity ZFN pair, designated N2, targeting intron 1 of the hF9 gene,shown below in Table 1 (also see United States Publication No.20110027235):

TABLE 1 hFactor9-specific ZFN pair N2 ZFN Name Target site F1 F2 F3 F4F5 SBS#9802 QSGDLTR RSDVLSE DRSNRIK RSDNLSE QNATRINtgACACAGTACCTGGCAccatagttgta (SEQ ID (SEQ ID   (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 3) NO: 4) NO: 5) NO: 6 NO: 7) NO: 8) SBS#11004 RSDSLSVTSGHLSR RSDHLSQ HASTRHC N/A gtACTAGGGGTATGgggataaaccagac (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 9) NO: 10) NO: 11 NO: 12) NO: 13)

The N2 ZFN expression plasmid for cell culture transfection wasconstructed by inserting a 2A linker from the Thosea asigna virusbetween the coding sequences for both ZFNs, and inserting this cassettedownstream of a CMV promoter. Upon transfection of the N2 ZFN pair intohuman K562 cells, we found that 30% of N2 target site alleles werecleaved (FIG. 1C). To test the ability of the N2 ZFNs to stimulatehomologous recombination by inducing DSBs, we co-transfected the N2 ZFNsinto K562 cells with a targeting vector that inserts a NheI restrictionsite (FIG. 1D). The NheI donor plasmid was constructed by amplifying theleft and right arms of homology from K562 genomic DNA by PCR. A shortsequence containing a NheI restriction site was subsequently introducedbetween the left and right arms of homology. We found that at days 3 and10 post-transfection, 14% and 12%, respectively, of alleles weresensitive to NheI digestion (FIG. 1E), indicative of efficient rates oftargeted integration (TI) through homologous recombination.

Example 2 In Vivo Murine Model of Human Hemophilia

The N2 target site is present in hF9 intron 1, but absent from themurine F9 gene. Thus, to test the N2 ZFNs in vivo we generated ahumanized mouse model of hemophilia B (HB). We constructed an hF9mini-gene (FIG. 2A) under control of a liver-specific promoter (Shen etal, (1989) DNA 8: 101-108 and Miao et al, (2000) Mol. Ther. 1: 522-532).Primers for TI of hF9 intron 1 were N2 TI For (GGCCTTATTTACACAAAAAGTCTG,SEQ ID NO:14) and N2 TI Rev (TTTGCTCTAACTCCTGTTATCCATC, SEQ ID NO:15).

Intron 1 of this construct contains the human N2 target site (LandingPad, LP). The remainder of the F9 sequence in the LP construct mimics apreviously identified nonsense mutation (Y155stop) (Thompson et al,(1989) Hum. Genet. 94: 299-302) in which a premature stop codon prior toexons encoding the FIX catalytic domain results in an absence ofcirculating FIX protein. The LP construct was constructed by genesynthesis (Genscript) and ligated into the pUC57 plasmid. The LPconstruct was then excised by SwaI digestion and ligated into the SwaIsite of a proprietary plasmid between FLP recombinase sites compatiblefor recombinase-mediated cassette exchange (RCME) (Taconic-Artemis) tocreate the LP KI plasmid. The LP KI plasmid and a FLP recombinaseexpression plasmid (Taconic-Artemis) were transfected into B6S6F1embryonic stem (ES) cells containing FLP recombinase sites compatiblefor RCME at the ROSA26 locus (Zambrowicz et al, (1997) Proc Natl AcadSci 94: 3789-3794). Correctly targeted B6S6F1-LP ES cell clones wereidentified by Southern blot and injected into B6D2F1 blastocysts. PureES cell derived B6S6F1-LP mice (G0) were delivered by natural birth, andchimeric pups were back-crossed with wild type C57BL/6J mice (JacksonLaboratories) for 5 generations (in vivo cleavage experiments) or 7-10generations (in vivo TI experiments).

LP mice were genotyped using primers LP Oligo 1 (ACTGTCCTCTCATGCGTTGG,SEQ ID NO:16), LP Oligo 2 (GATGTTGGAGGTGGCATGG, SEQ ID NO:17), wtROSAOligo 1 (CATGTCTTTAATCTACCTCGATGG, SEQ ID NO:18), and wtROSA Oligo2(CTCCCTCGTGATCTGCAACTCC, SEQ ID NO:19) (FIG. 2B). We also crossed LPmice with an existing mouse model that has a deletion of the murine F9gene (Lin et al, (1997) Blood 90, 3962-3966) to generate LP/HB mice inwhich we could test N2 ZFN activity in vivo.

HB mice have been back-crossed with C57BL/6J mice (Jackson Laboratories)for >10 generations. C57BL/6J mice (Jackson Laboratories) were used forLP-negative TI experiments. As expected, LP mice did not have detectablecirculating hFIX (FIG. 2 c). Quantification of plasma hFIX was performedusing an hFIX ELISA kit (Affinity Biologicals), with a standard curvefrom pooled normal human plasma (Trinity Biotech). All values below thelast value of the standard curve (15 ng/mL) were arbitrarily given thevalue of 15 ng/mL, which is the limit of detection. Plasma for hFIXELISA was obtained by retro-orbital bleeding into heparinized capillarytubes.

Example 3 Targeted Delivery of FIX-Specific ZFNs In Vivo

To deliver the N2 ZFNs to the liver, the normal site of FIX production,we generated a hepatotropic adeno associated virus vector, serotype 8(AAV8-N2) expressing the N2 ZFNs from a liver-specific enhancer andpromoter (Shen et al, ibid and Miao et al, ibid) (FIG. 2D).

To test the cleavage activity of the N2 ZFNs in vivo we performed tailvein injections into LP mice using 1e11 v.g. AAV8-N2 expression vectorand isolated liver DNA at day 7 post-injection. PCR-amplification of theLP and Cel-I assay demonstrated that 34-47% of LP alleles had beencleaved (FIG. 2E). Primers for Cel-I of the LP construct were LP N2 For(CTAGTAGCTGACAGTACC, SEQ ID NO:20) and LP N2 Rev(GAAGAACAGAAGCCTAATTATG, SEQ ID NO:21).

Example 4 In Vivo Co-Delivery of a Donor Nucleic Acid and Fix-SpecificZFNs

Insertion of the wild-type exons 2-8, preceded by a splice acceptor(SA), into intron 1 of the LP construct allows for splicing of wild typecoding sequence with exon 1 (FIG. 3A). To correct the mutated F9 gene insitu in LP mice, we generated an AAV donor template vector (AA V-Donor)for gene targeting, with arms of homology, flanking a “SA—wild-type hF9exons 2-8” cassette (FIG. 3A). The donor vector production plasmids wereconstructed by amplifying the left and right arms of homology from LPmouse genomic DNA by PCR. The “splice acceptor—exons 2-8 codingsequence—bovine growth hormone polyA signal” cassette was obtained byPCR amplification from the pAAV-hFIX16 plasmid (Manno et al, (2006) Nat.Med. 12: 342-347) and ligated between the left and right arms ofhomology. Since HR is favored during the S/G2 phases of the cell cycle,we delivered the N2 and Donor vectors to neonatal mice, where rapidlyproliferating hepatocytes enter S/G2 during cell cycle progression.

We injected LP/HB mice at day 2 of life with either AAV-N2 (5e10 v.g)alone (n=1), AAV-N2 (5e10 v.g)+AAV-Donor (2.5e11 v.g) (n=5), or AAV-Mock(5e10 v.g)+AAV-Donor (2.5e11 v.g) (n=5). In the Mock vector, the N2 ZFNshave been replaced by renilla luciferase.

At week 10 of life, we sacrificed mice and isolated liver DNA to assayfor TI of the donor using two separate PCR strategies. The firststrategy uses primers that generate a smaller amplicon for a targeted LPallele and a larger amplicon for an untargeted LP allele (FIG. 3 a,primers P1/P2). The second strategy involves using primers that generatea larger amplicon for a targeted LP allele and a smaller amplicon for anuntargeted LP allele (FIG. 3A, primers P1/P3). Primers for TI of the LPconstruct were P1 (ACGGTATCGATAAGCTTGATATCGAATTCTAG, SEQ ID NO:22), P2(CACTGATCTCCATCAACATACTGC, SEQ ID NO:23), and P3(GAATAATTCTTTAGTTTTAGCAA, SEQ ID NO:24).

When we performed both PCR analyses, we found evidence of TI only inmice receiving N2+Donor (FIG. 3B). Quantification of band intensitiessuggested 1-7% TI frequency (FIG. 3B).

To determine if genomic correction results in production of circulatinghFIX, we injected LP mice at day 2 of life with AAV-N2 alone (n=7),AAV-Mock+AAV-Donor (n=6), or AAV-N2+AAV-Donor (n=7) (same vector dosesas above). Plasma hFIX levels for mice receiving N2 alone or Mock+Donoraveraged less than 15 ng/mL (the lower limit of detection of the assay),while mice receiving N2+Donor averaged 116-121 ng/mL (corresponding to2-3% of normal) (FIG. 4A), significantly greater than mice receiving N2alone and Mock+Donor (p≦0.006 at all time points, 2-tailed T-test).

To confirm stable genomic correction, we performed partial hepatectomies(PHx), which cause extra-chromosomal episomes to be diluted and lost ashepatocytes proliferate during liver regeneration (Nakai et al, (2001),J. Virol. 75: 6969-6976) (FIG. 4 A,B). Partial hepatectomies wereperformed as previously described (Mitchell and Willenbring, (2008) Nat.Prot. 3:1167-1170) and all animal procedures were approved by theChildren's Hospital of Philadelphia IACUC. Measurement of hFIX levels inN2+Donor-treated mice were unchanged following liver regenerationpost-hepatectomy, indicating stable correction. Control mice receivingN2 alone or Mock+Donor continued to average less than 15 ng/mL (FIG. 4A)post-hepatectomy, significantly lower than mice receiving N2+Donor(p≦0.004 at all time points, 2-tailed T-test). To ensure hFIX expressionwas LP-specific and did not result from random donor integration intothe genome, we injected wild-type mice lacking the LP transgene at day 2of life with AAV-N2 alone (n=8), AAV-Mock+AAV-Donor (n=6), orAAV-N2+AAV-Donor (n=9) (same vector doses as above). Plasma hFIX levelsfor mice receiving N2 alone, Mock+Donor, and N2+Donor averaged less than15, 19, and 27 ng/mL, respectively, indicating the majority of hFIXexpression in LP mice receiving N2+Donor came from LP-specificcorrection (FIG. 4C).

To determine if hFIX levels were sufficient to correct the HB phenotype,we injected LP/HB mice at day 2 of life with AAV-N2 alone (n=10),AAV-Mock+AAV-Donor (n=9), or AAV-N2+AAV-Donor (n=9) (same vector dosesas above). Plasma hFIX levels for mice receiving N2 alone again averagedless than 15 ng/mL. Mice receiving Mock+Donor averaged less than 25ng/mL, and mice receiving N2+Donor had significantly higher hFIX levels(p≦0.04 at all time points compared to Mock+Donor, 2-tailed T-test),averaging 166-354 ng/mL (3-7% of normal circulating levels) (FIG. 4D).We confirmed liver-specific hFIX expression by RT-PCR for hFIX mRNA(FIG. 4E). RNA from frozen mouse tissue was isolated using the RNeasykit (Qiagen) and the RNase-free DNase kit (Qiagen). cDNA synthesis wasperformed using the iSCRIPT kit (Bio-Rad). RT-PCR for hFIX transcriptwas performed using primers hFIX Gen1 For (ACCAGCAGTGCCATTTCCA, SEQ IDNO:25) and hFIX Gen1 Rev (GAATTGACCTGGTTTGGCATCT, SEQ ID NO:26)

To assay whether the HB phenotype was corrected, we measured activatedpartial thromboplastin time (aPTT), a measure of kinetics of fibrin clotformation that is markedly prolonged in hemophilia. aPTT was performedby mixing sample plasma 1:1:1 with pooled HB human plasma and aPTTreagent. Clot formation was initiated by addition of 25 mM calciumchloride. Plasma for aPTT was obtained by tail bleeding 9:1 into sodiumcitrate. The average aPTTs for wild-type mice (n=5) and HB mice (n=12)were 36 seconds and 67 seconds, respectively (FIG. 4F). Mice receivingMock+Donor (n=3) averaged 60 seconds, while mice receiving N2+Donor(n=5) had a significantly shortened aPTT, averaging 44 seconds (p=0.0014compared to Mock+Donor, 2-tailed T-test) (FIG. 4F).

Example 5 In Vivo Co-Delivery of Engineered Nucleases and Donor in AdultAnimals

Adult animals were then subjected to genome editing at the human F.IX LPas described above for the neonates. Adult LP mice were treated by I.V.injection at 6 weeks with either 1e¹¹ v.g./mouse AAV-N2 alone (‘ZFNalone’), 1e¹¹ v.g./mouse AAV-N2 and 5.5e11 v.g./mouse AAV-Donor(‘ZFN+Donor’), or 1e¹¹ v.g./mouse AAV-Mock and 5.5e11 v.g. AAV-Donor(‘Mock+Donor’). The data depicted in FIG. 5A is representative of 3experiments with approximately 20 mice per group. In these experiments,the wild type hF.IX levels were approximately 1000 ng/mL. Similarly,FIG. 5B is a graph showing the plasma hFIX levels in adult LP micefollowing I.V. injection at 6 weeks of age with either 1e¹¹ v.g./mouseAAV-N2 alone (‘ZFN alone’), 1e¹¹ v.g./mouse AAV-N2 and 5.5e11 v.g./mouseAAV-Donor (‘ZFN+Donor’), or 1e¹¹ v.g./mouse AAV-Mock and 5.5e11 v.g.AAV-Donor (‘Mock+Donor’). Two days following injection, the groups inFIG. 5B were given a partial hepatectomy. The data depicted isrepresentative of 3 experiments with approximately 20 mice per group. Inthese experiments, the wild type hF.IX levels were approximately 1000ng/mL. The data demonstrate that hF.IX expression is stable when givento adult mice with or without a follow on partial hepatectomy, and thatit is possible to perform genome editing in adult animals.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

1. A protein comprising an engineered zinc finger protein DNA-bindingdomain, wherein the DNA-binding domain comprises four or five zincfinger recognition regions ordered F1 to F4 or F1 to F5 from N-terminusto C-terminus, and wherein (i) when the DNA-binding domain comprisesfive zinc finger recognition regions, F1 to F5 comprise the followingamino acid sequences: (SEQ ID NO: 4) F1: QSGDLTR (SEQ ID NO: 5)F2: RSDVLSE (SEQ ID NO: 6) F3: DRSNRIK (SEQ ID NO: 7) F4: RSDNLSE(SEQ ID NO: 8) F5: QNATRIN;

(ii) when the DNA-binding domain comprises four zinc finger recognitionregions, F1 to F4 comprise the following amino acid sequences:(SEQ ID NO: 10) F1: RSDSLSV (SEQ ID NO: 11) F2: TSGHLSR (SEQ ID NO: 12)F3: RSDHLSQ (SEQ ID NO: 13) F4: HASTRHC.


2. The protein according to claim 1, further comprising a cleavagedomain or cleavage half-domain.
 3. The protein of claim 2, wherein thecleavage half-domain is a wild-type or engineered FokI cleavagehalf-domain.
 4. A polynucleotide encoding the protein of claim
 1. 5. Agene delivery vector comprising a polynucleotide according to claim 4.6. An isolated cell comprising the protein of claim
 1. 7. An isolatedcell comprising the polynucleotide of claim
 4. 8. A method for treatinghemophilia B in a subject, the method comprising integrating a sequenceencoding a functional Factor IX (FIX) protein into a FIX gene in thegenome of a cell using at least one zinc finger nuclease according toclaim 1, wherein the subject comprises the cell. 9-10. (canceled) 11.The method of claim 8, wherein the sequence is delivered to the cellusing a vector selected from the group consisting of a viral vector, anon-viral vector and combinations thereof.
 12. The method of claim 8,wherein the at least one zinc finger nuclease is delivered to the cellusing a vector selected from the group consisting of a viral vector, anon-viral vector and combinations thereof.
 13. The method of claim 8,wherein the cell is a hepatic cell and the sequence is delivered to thecell by intravenous administration into the liver of an intact animal,intraperitoneal administration, direct injection into liver parenchyma,injection into the hepatic artery, or retrograde injection through thebiliary tree.
 14. The method of claim 8, further comprising the step ofperforming a partial hepatectomy on the subject.
 15. The method of claim8, further comprising the step of treating the subject with at least onesecondary agent.
 16. The method of claim 15, wherein the secondary agentis selected from the group consisting of gamma irradiation, UVirradiation, tritiated nucleotides, cis-platinum, etoposide,hydroxyurea, aphidicolin, prednisolone, carbon tetrachloride, adenovirusand combinations thereof.
 17. The method of claim 8, wherein the cell isan isolated cell and the method further comprises administering theisolated cell to the subject.
 18. The method of claim 8, wherein thesubject is selected from the group consisting of an embryo, a fetus, aneonate, an infant, a juvenile or an adult.
 19. The method of claim 8,further comprising associating the sequence with a homing agent thatbinds specifically to a surface receptor of the cell.
 20. The method ofclaim 19, wherein the homing agent comprises galactose or a hybrid of anAAV coat protein and galactose.
 21. The method of claim 8, furthercomprising associating a polynucleotide encoding the at least onenuclease with a homing agent that binds specifically to a surfacereceptor of the cell.
 22. The method of claim 21, wherein the homingagent comprises galactose or a hybrid of an AAV coat protein andgalactose.
 23. The method of claim 8, wherein the cell is selected fromthe group consisting of a human cell, a nonhuman primate cell, a Rodentacell, a Lagomorpha cell, a Carnivora cell and an Arteriodactyla cell.24. The method of claim 8, wherein the cell is a stem cell.
 25. Themethod of claim 24, wherein the stem cell is an embryonic stem cell, ahematopoietic stem cell, an induced pluripotent stem cell, a hepatocyteor a hepatic stem cell.